The present invention relates generally to procedures for preparing biologically active polypeptide products and more particularly to procedures for establishing a biologically active natural conformation in polypeptides containing multiple disulfide bonds.
In the recent past substantial advances have been made in recombinant DNA procedures for securing synthesis of a wide variety of polypeptide products in microorganisms, including bacteria, yeast and mammalian cells in culture. "Foreign" polypeptides can now rather readily be expressed in relatively high yields in genetically transformed host cell cultures, either as discrete polypeptides or as fusion polypeptides including the desired polypeptide linked in sequence to a second, usually endogenous, polypeptide such as .beta.-galactosidase.
These advances in securing expression of polypeptides have given rise to an entire new series of problems relating to recovery of desired polypeptide products from microorganisms in useful forms. Apart from the general difficulties inherent in isolating polypeptide products away from native host cell proteins, recovery problems are particularly significant in microbial systems employed for synthesis of biologically active polypeptides such as human growth hormone, interferons and the like, where the biological activity (and potential utility) of the recovered polypeptide product is dependent upon the product's assumption of secondary and tertiary structural conformations duplicative of naturally occurring forms.
A polypeptide exists as a chain of amino acids linked by peptide bonds. In the normal biologically active form of a polypeptide (hereinafter referred to as the native form) one or more chains are folded into a thermodynamically preferred three dimensional structure, the conformation of which is determined by steric considerations, the existence of covalent disulfide bonds, if any, and noncovalent interatomic forces such as charge influences, hydrogen bonding and hydrophobic interactions. The amino acid cysteine contains a sulfur atom which is capable of forming cystine disulfide bonds with other cysteine residues. These bonds are capable of forming on an intra- or interchain basis and play a key role in establishing a stable tertiary structure for many polypeptides.
In the isolation of polypeptide products from recombinant host cells, it is often the case that recovered polypeptides fail to adopt their native conformation and, as a result, are biologically inactive with respect to certain desired properties. It is thought that this occurs because the cellular environment of the recombinant host often does not provide conditions in which the proper "folding" of the foreign polypeptide can spontaneously occur after synthesis. However, since it is a generally accepted tenet that information dictating the tertiary structure of polypeptides resides in their primary sequence, such inactive polypeptides can occasionally be induced to adopt their biologically active "native" structure by a simple denaturation and renaturation process. A denaturing agent can be added to a polypeptide which will disrupt the noncovalent interatomic forces and effectively "unfold" the molecule. The polypeptide can then be renatured by removal or dilution of the denaturing agent so that the polypeptide adopts its native conformation.
In many cases, however, biologically inactive polypeptides represent molecules which are "frozen" in a non-native conformation because of the formation of non-native cystine disulfide bonds. These bonds frequently occur during polypeptide expression in host organisms before the polypeptide molecule adopts its favored native conformation. With increasing numbers of cysteine residues in a polypeptide, the probability that disulfide bonds will properly form decreases exponentially. Once non-native bonds form, however, the polypeptide is effectively locked out of its native conformation. Because disulfide bonds are covalent, polypeptides possessing non-native structures as a result of incorrect cystine bond formation are not readily susceptible to reformation by a simple denaturation/renaturation procedure.
The difficulties associated with recovery of biologically active polypeptides containing multiple disulfide bonds have been so severe that in a few instances new polypeptide analogs of significant proteins have been "designed" for microbial expression on the basis of their greater potential for recovery in a biologically active state rather than for possibly enhanced or prolonged activity. As one example, the general inability to recover a beta interferon polypeptide (three cysteine residues) in biologically active form prompted construction of genes for expression of various des-cysteine analogs wherein undesired disulfide bond formation was precluded by limiting the number of cysteine residues available for such reactions from three to two. Mark, et al., "The Effects of Site Specific Mutation on the Biological Activity of Human Fibroblast Interferon", Abstract, Second International T.N.O. Meeting on the Biology of the Interferon System, Antiviral Research (1983). For polypeptides with two or more cystine bonds, however, such techniques will be limited in their effectiveness.
Workers have utilized several methods to reform native conformations in multiple disulfide bond polypeptides. The simplest of these involve techniques whereby a polypeptide molecule is denatured, its cystine disulfide bonds are reductively cleaved, native disulfide bonds are allowed to form and the polypeptide is renatured to its native conformation. Stryer, Biochemistry, 32-36 (2d Ed. 1981), describes work on denaturing and reforming the native conformation of ribonuclease, a protein with a single polypeptide chain consisting of 124 amino acid residues and having four disulfide bonds. It was noted that natural ribonuclease in its properly folded form could be denatured by exposure to a concentrated urea solution in the presence of a reducing agent such as .beta.-mercaptoethanol which cleaves the four disulfide bonds to yield eight cysteine residues. The denatured compound was completely uncoiled and exhibited no enzymatic activity. The reduced ribonuclease solution was dialyzed allowing the urea and reducing agent to diffuse away from the polypeptide. Upon air oxidation of the reduced cysteine residues in the ribonuclease, the polypeptide spontaneously refolded into its native conformation and the native disulfide bonds reformed as determined by a return of catalytic activity.
Other methods are also known for establishing native conformations in microbiologically-produced disulfide containing polypeptides. Lowe, et al. U.K. patent application No. 2,138,004A discloses variations on the above process whereby polypeptides may be denatured and their disulfide bonds reduced in the presence of denaturing agents such as guanidine hydrochloride or urea used in combination with an alkali reducing agent. Guanidine hydrochloride and urea are capable of denaturing a polypeptide chain but are incapable of cleaving disulfide bonds. Conversely, a strong alkali solution is capable of reductively dissociating disulfide bonds but may not alone be capable of completely denaturing some polypeptides. According to the procedure of Lowe, et al., a polypeptide is first denatured in an aqueous solution with guanidine hydrochloride or urea and is then diluted in an alkaline aqueous solution at a pH selected to promote dissociation of the group or groups of the polypeptide involved in maintaining the non-native conformation of the polypeptide. The polypeptide is then renatured by reducing the pH of the solution below a pH effective to denature the polypeptide to produce the native conformation. The method is disclosed to produce satisfactory results when applied to the protein prochymosin, a monomeric protein containing three intramolecular disulfide bonds. Reduction of the cystine bonds can be achieved by using an alkaline aqueous solution of pH 10.7 (.+-.0.5) as these workers state that the free thiol groups of cysteine in prochymosin have a pKa value of 10.46.
Use of pH based procedures for denaturation and renaturation of polypeptides is not without its limitations, however. Extreme low or high pHs can irreversibly denature polypeptides by reacting with amino acid residues making up the structure. Care must also be taken that denatured and reduced polypeptides do not prematurely reoxidize and renature. Even if a polypeptide is completely denatured and its disulfide bonds completely reduced, it is possible that cysteine residues may spontaneously reoxidize to form non-native conformations. This can be a particular problem where the polypeptide solution is a fairly concentrated one and reactive cysteine residues can easily associate with cysteine residues from other molecules to form dimers.
Some workers have sought to prevent spontaneous non-native bonding by forming intermediate adducts with the reduced cysteine residues. The reduced cysteine residues are reacted in an oxidation reaction with a disulfide containing compound which prevents the cysteine residue so reacted from reacting with other cysteine residues on the same or other polypeptide molecules. The polypeptide solution may then be diluted into a buffer containing a weak reducing agent, the disulfide group intermediates reduced and the polypeptide molecules allowed to slowly renature to their native conformation. The fact that the polypeptide molecules are "protected" from reaction until they are diluted, substantially raises the probability that the polypeptides will renature to their native conformation.
Among intermediate forming techniques known in the art, Wetzel, et al., Gene, 16, pp. 63-71 (1981), describes efforts directed toward purification of a "mini-C proinsulin" product from a culture system involving E. coli expression of a .beta.-galactosidase fusion protein. The fusion polypeptide was harvested and was treated with cyanogen bromide to cleave the proinsulin product from the fusion protein. The cleaved proinsulin product was solubilized with guanidine hydrochloride denaturing agent and treated with sodium sulfite and sodium tetrathionate reducing agents in order to form a stable "S-sulfonate" oxidized reaction product at the site of the cysteine residues in the polypeptide. The resulting mini-C proinsulin S-sulfonate product was thereafter further processed with a .beta.-mercaptoethanol reducing agent at 0.degree. C. and under a nitrogen atmosphere to dissociate the covalently-bound sulfonate groups. This prompted formation of native disulfide bonds and allowed assumption of the tertiary structure needed for achievement of biological activity.
Numerous variations on the above-mentioned techniques are also known in the art. Builder, et al. U.S. Pat. No. 4,511,502, Olson, et al. U.S. Pat. No. 4,511,503 and Jones, et al., U.S. Pat. No. 4,512,922 disclose the use of "strongly denaturing solutions" comprising guanidine hydrochloride or sodium thiocyanate in high concentrations of approximately 4-9M or detergents such as sodiumdodecyl sulfate (SDS) or Triton-X-100 in concentrations of about 0.01 to about 2%. Also disclosed are "weakly denaturing solutions" comprising either urea or the materials of strongly denaturing solutions at lesser concentrations. Among the reducing agents disclosed are .beta.-mercaptoethanol, dithiothreitol and reduced glutathione. Compounds disclosed for disulfide adduct formation include oxidized glutathione, cystamine and cystine. A disulfide adduct forming renaturation technique is disclosed whereby a polypeptide is denatured in a strong denaturing solution containing a reducing agent which reductively dissociates any disulfide bonds. The polypeptide is then treated with a mild oxidizing agent in the presence of sulfite ion to form disulfide adducts. The strong denaturing solution is then replaced with a weakly denaturing solution to permit refolding, and disulfide linkages are reformed using sulfhydryl compounds such as, for example, cysteine or reduced glutathione, in the presence of the corresponding oxidized (disulfide) form, but with the reduced form in excess.
Also known is a "simultaneous" unfolding and refolding procedure whereby a polypeptide is placed into a sulfhydryl/disulfide-containing buffer, which buffer has sufficient denaturing power that all of the intermediate conformations remain soluble in the course of unfolding and refolding. Both reduced and oxidized (disulfide) forms of sulfhydryl compounds are disclosed to be in the medium. In this redox buffer refolding procedure, the molar ratio of reduced to oxidized forms of sulfhydryl compounds is disclosed to be from 5:1 to 20:1. The pH must be sufficiently high so as to assure at least partial ionization of the sulfhydryl groups but not so high as to irreversibly denature the polypeptide.
In recent years much work has been directed toward recombinant microbial synthesis of the extremely sweet polypeptide thaumatin. Thaumatin is produced in the arils of the fruit of the African shrub Thaumatococcus daniellii Benth. The fruit traditionally has been used in West Africa as a sweetener of palm wine, corn, bread and sour fruit. Thaumatin, which is about 5000 times sweeter than sucrose on a weight basis, is produced in at least five forms: thaumatins I, II, a, b and c. These polypeptides, named in their order of elution from an ion exchange column [Higgenbotham, et al., in Sensory Properties of Foods (Birch, et al., eds.), London: Applied Sciences, pp. 129-149 (1977)], have molecular weights of approximately 22 kilodaltons.
Thaumatins I and II are non-toxic polypeptides, are low-calorie and non-cariogenic, and elicit profound sweet taste responses suggesting a stable interaction between these polypeptides and human taste buds. Therefore, thaumatin has potential for use as a sugar substitute, food additive, a sweetness receptor probe and a tool for further elucidation of the taste response.
A plentiful supply of pure thaumatin is required to utilize the protein as a possible food additive and research tool. Because the thaumatin plant requires a tropical climate and insect pollination for successful fruit propagation, there are considerable difficulties involved in greenhouse cultivation of the fruit. For these reasons, considerable effort has been directed toward the introduction of genes into recombinant microorganisms enabling them to synthesize thaumatin. One research group has reported the successful cloning of a gene for thaumatin II from messenger RNA-derived cDNA [Edens, et al., Gene, 18, 1-12 (1982)]. The Edens, et al. reference cited above notes that a polypeptide having the native sequence of preprothaumatin II has been microbially produced. More specifically, the reference and European patent application Nos. 54,330 and 54,331 disclose cDNA sequences coding for native mature thaumatin II and preprothaumatin II and also disclose cloning vehicles comprising the DNA sequences for use in transformation in microorganisms.
In co-owned and copending U.S. patent application Ser. No. 540,634 filed Oct. 11, 1983, now abandoned, the successful synthesis of "manufactured" genes coding for thaumatin I having a primary structural conformation duplicating the sequence provided in Iyengar, et al. Eur. J. Biochem., 96, 193-204 (1979) was disclosed along with their expression in bacterial and yeast hosts. The polypeptides that have been expressed contain the primary conformation (amino acid sequence) of thaumatin I but are not always sweet and often do not exhibit the secondary and tertiary conformations of the native polypeptide. It is believed that similar difficulties plague other workers in the field.
The failure of the recombinant-produced thaumatin to adopt the native conformation and activity of plant produced thaumatin is believed to result from the different cellular milieu found in the recombinant organism. The solubility, pH and electronic environment of the recombinant host cell is such that the thaumatin adopts a conformation lacking biological activity. Conventional methods for establishing the native conformation and biological activity demonstrate only limited success. A complex globular structure postulated for the thaumatin molecule is disclosed in de Vos, et al., Proc. Natl. Acad. Sci., 82, 1406 (1985), which shows 16 cysteine residues combining to form 8 specific disulfide bonds. This large number of cystine bonds makes folding to the native structure extremely difficult.
The difficulty in establishing native conformations in thaumatin and other polypeptides containing high numbers of disulfide bonds when using traditional adduct forming techniques, stems, at least partially, from premature reoxidation of cysteine residues to form non-native disulfide bonds during the adduct formation step. Between the step of denaturing the polypeptide and reducing any existing disulfide bonds and the step of introducing disulfide group containing compounds to form the disulfide adduct, the concentration of the reducing agent is often reduced in conventional adduct forming methods. During the period while the concentration of the reducing agent is being reduced and before intermediate adducts have been formed on the reduced cysteine residues, many of the cysteine residues will spontaneously oxidize with other cysteine residues to form non-native disulfide bonds. Once such incorrect bonds have been formed, they may preclude the polypeptide from assuming its correct conformation.
Accordingly, there exists a need in the art for techniques for generating biologically active native conformations of polypeptides, especially recombinant-produced polypeptides with multiple cysteine residues. The techniques should be efficient, producing a high yield of polypeptide in its correct conformation and should be relatively rapid.